Transcript Slide 1

Basic Instrumentation
Joachim Mueller
Principles of Fluorescence Spectroscopy
Genova, Italy
June 19-22, 2006
Figure and slide acknowledgements:
Theodore Hazlett
ISS PC1 (ISS Inc., Champaign,
IL, USA)
Fluorolog-3 (Jobin Yvon Inc, Edison,
NJ, USA )
QuantaMaster (OBB Sales, London,
Ontario N6E 2S8)
Fluorometer Components
Excitation
Polarizer
Sample
Light Source
Excitation Wavelength
Selection
Emission
Polarizer
Emission
Wavelength
Selection
Computer
Detector
Fluorometer: The Basics
Note: Both polarizers can be removed from the
optical beam path
Fluorometer Components
Light Source
Detectors
Wavelength Selection
Polarizers
The Laboratory Fluorometer
Standard Light Source:
Xenon Arc Lamp
Exit Slit
Pex
Pem
Pem
ISS (Champaign, IL, USA) PC1 Fluorometer
Light Sources
Xenon Arc Lamp Profiles
Lamp Light Sources
1. Xenon Arc Lamp (wide
range of wavelengths)
2. High Pressure Mercury Lamps
(High Intensities but
concentrated in specific lines)
Ozone Free
Visible
UV
3. Mercury-Xenon Arc Lamp
(greater intensities in the UV)
4. Tungsten-Halogen Lamps
5. Light emitting diodes (LEDs)
Multiple color LEDs can be
bunched to provide a broad
emission range)
Mercury-Xenon Arc Lamp Profile
Light Emitting Diodes (LED)
Wavelengths from
350 nm to 1300 nm
Near UV
LED
Lasers Light Sources
528nm
514nm 532nm
543nm
Titanium:Sapphire
633nm 690 nm – 990 nm
442nm
325nm
488nm
295nm
200
300
Argon-ion
100 mW
351 nm
364 nm
400
Helium-cadmium
576nm
500
Nd-YAG
600
Green
He-Ne
10 mW
700
Wavelength (nm)
Orange
He-Ne
10 mW
He-Ne
>10 mW
Laser Diodes
300
400
500
Wavelength (nm)
600
700
Detectors
Scallop
Scallop
Eyes
From http://www.eyedesignbook.com/index.html
Image courtesy of BioMEDIA ASSOCIATES http://www.ebiomedia.com
APD
The silicon avalanche photodiode (Si APD) has
a fast time response and high sensitivity in the
near infrared region. APDs can be purchased
from Hamamatsu with active areas from 0.2
mm to 5.0 mm in diameter and low dark
currents (selectable). Photo courtesy of
Hamamatsu
MCP & Electronics
(ISS Inc. Champaign, IL USA)
The Classic PMT Design
l
Dynodes
Vacuum
Photocathode
e-
e-ee
e-e--e e -eeeee-
Anode
Window
Constant Voltage
(use of a Zenor Diode)
High Voltage Supply
(-1000 to -2000 V)
Current Output
resister series
(voltage divider)
Ground
capacitor series
(current source)
Hamamatsu R928 PMT Family
R2949
Window with
Photocathode Beneath
PMT Quantum Efficiencies
Cathode Material
Window Material
Photon Counting (Digital) and Analog Detection
Signal
time
Continuous
Current Measurement
Analog:
Photon Counting:
Variable
Voltage Supply
Constant
High Voltage Supply
PMT
PMT
Discriminator
Sets Level
Anode Current
=
Pulse averaging
level
TTL Output
(1 photon = 1 pulse)
Computer
Primary Advantages:
1. Sensitivity (high signal/noise)
2. Increased measurement stability
Primary Advantage:
1. Broad dynamic range
2. Adjustable range
Wavelength Selection
Fixed Optical Filters
Tunable Optical Filters
Monochromators
Optical Filter Channel
Pex
Pem
Pem
Long Pass Optical Filters
Transmission (%)
100
80
Spectral Shape
Thickness
Physical Shape
Fluorescence (!?)
60
40
20
0
300
400
500
600
Wavelength (nm)
Hoya O54
700
800
More Optical Filter Types…
Interference Filters
(Chroma Technologies)
Broad Bandpass Filter
(Hoya U330)
100
Transmission (%)
80
60
40
20
0
300
400
500
600
700
Wavelength (nm)
Neutral Density
(Coherent Lasers)
Tunable Optical Filters
Liquid Crystal Filters:
An electrically controlled liquid crystal elements to select a specific visible wavelength of
light for transmission through the filter at the exclusion of all others.
AO Tunable Filters:
The AOTF range of acousto-optic devices are solid state optical filters. The wavelength
of the diffracted light is selected according to the frequency of the RF drive signal.
Isomet (http://www.isomet.com/index.html)
Monochromators
Mirrors
Czerny-Turner design
1. Slit Width (mm) is the
dimension of the slits.
2. Bandpass is the FWHM of
the selected wavelength.
Exit Slit
3. The dispersion is the factor
to convert slit width to
bandpass.
Entrance slit
Rotating Diffraction Grating
(Planar or Concaved)
The Inside of a Monochromator
Mirrors
Grating
Nth Order
(spectral distribution)
Zero Order
(acts like a mirror)
Changing the Bandpass
1. Drop in intensity
2. Narrowing of the spectral selection
Fixed Excitation Bandpass =
4.25 nm
Changing the Emission Bandpass
1.0
0.8
17 nm
0.6
8.5 nm
0.4
4.25 nm
6
(au)
Fluorescence
x10
1.0
2.125 nm
0.2
17 nm
0.8
0.6
8.5 nm
0.4
4.25 nm
0.2
2.125 nm
0.0
0.0
520
540
560
Wavelength (nm)
580
520
540
560
Wavelength (nm)
Collected on a SPEX Fluoromax - 2
580
Higher Order Light Diffraction
Emission Scan:
Excitation 300 nm
Glycogen in PBS
350
300
3
(au)
Fluorescence
x10
2nd Order Scatter
(600 nm)
Excitation (Rayleigh) Scatter
(300 nm)
250
200
2nd Order RAMAN
(668 nm)
Water RAMAN
(334 nm)
150
100
50
0
200
300
400
500
Wavelength (nm)
600
700
Fluorescent Contaminants
Monochromator Polarization Bias
Tungsten Lamp Profile Collected on an SLM Fluorometer
Wood’s Anomaly
Parallel Emission
250
Fluorescence
Fluorescence
No Polarizer
Perpendicular Emission
800
250
Adapted from Jameson, D.M., Instrumental Refinements in Fluorescence
Spectroscopy: Applications to Protein Systems., in Biochemistry,
Champaign-Urbana, University of Illinois, 1978.
800
Correction of Emission Spectra
ISSPC1
Correction Factors
300
350
400
vertical
horizontal
450
500
550
600
Wavelength (nm)
Wavelength
ANS Emission Spectrum, no polarizer
ANS Emission Spectrum, parallel polarizer
C
corrected
Fluorescence
Intensity (a.u.)
Fluorescence
Intensity (a.u.)
B
uncorrected
400
450
500
Wavelength (nm)
Wavelength
550
600
400
450
500
550
600
Wavelength (nm)
Wavelength
from Jameson et. Al., Methods in Enzymology, 360:1
Excitation Correction
Quantum Counter
Exit Slit
Pex
Pem
Pem
The Instrument Quantum Counter
Common Quantum Counters
(optimal range)*
Eppley Thermopile/ QC
Optical Filter
Rhodamine B
(220 - 600 nm)
Fluorescein
(240 - 400 nm)
Quinine Sulfate
(220 - 340 nm)
Quantum Counter
Reference
Detector
1.2
0.8
0.4
0.0
200
Linearity of Rhodamine
as a quantum counter
Fluorescence
Here we want the inner filter effect!
400
600
Wavelength (nm)
* Melhuish (1962) J. Opt. Soc. Amer. 52:1256
Excitation Correction
Absorption (dotted line) and Excitation Spectra (solid line) of ANS in Ethanol
1.0
1.0
Ratio Corrected
0.8
B
Fluorescence
0.8
0.6
0.4
0.2
0.0
0.6
0.4
0.2
0.0
250
300
350
400
450
250
300
Wavelength (nm)
Wavelength
350
400
450
Wavelength (nm)
Wavelength
1.0
C
Fluorescence
Fluorescence
A
Uncorrected
Lamp
Corrected
0.8
0.6
0.4
0.2
0.0
250
300
350
400
450
Wavelength (nm)
Wavelength
from Jameson et. Al., Methods in Enzymology, 360:1
Polarizers
The Glan Taylor prism polarizer
Two Calcite Prisms
0
90
0
Common Types:
Glan Taylor (air gap)
Glan Thompson
Sheet Polarizers
90
Two UV selected calcite prisms are
assembled with an intervening air space. The
calcite prism is birefringent and cut so that only
one polarization component continues straight
through the prisms. The spectral range of this
polarizer is from 250 to 2300 nm. At 250 nm
there is approximately 50% transmittance.
Sample Issues
Signal Attenuation of the Excitation Light
PMT Saturation
Excess Emission
30
Fluorescence vs Signal
3
6
x10
2
20
6
LINEAR REGION
15
1
10
540
x10
Instrument Signal
25
560
580
600
620
640
660
Wavelength (nm)
[Fluorophore]
Reduced emission intensity
1. ND Filters
2. Narrow slit widths
3. Move off absorbance peak
680
700
Attenuation of the Excitation Light through Absorbance
Sample concentration
& the inner filter effect
Rhodamine B
from Jameson et. al., Methods in Enzymology (2002), 360:1
The second half of the inner filter effect:
attenuation of the emission signal.
1.0
4
3
Diluted Sample
3
0.8
6
x10
2
2
0.4
1
1
1
0.2
450
500
550
600
Wavelength (nm)
Absorbance Spectrum
650
700
540
560
580
600
620
640
Wavelength (nm)
(1) Spectral Shift
(2) Change in Spectral Shape
660
6
x10
0.6
2
x10
6
3
How do we handle highly absorbing solutions?
Quartz/Optical Glass/Plastic Cells
Excitation
Emission
Emission
Path Length
4 Position Turret
SPEX Fluoromax-2, Jobin-Yvon
Detector
Excitation
Path Length
Front Face Detection
Thin Cells & Special Compartments
Triangular Cells
IBH, Glasgow G3 8JU
United Kingdom
Excitation
Excitation
Emission
Detector
Sample
[1]
Absorbance
Measurements
Reflected Excitation & Emission
[1] Adapted from Gryczynski, Lubkowski, & Bucci Methods of Enz. 278: 538
Lifetime Instrumentation
Light Sources for Decay Acquisition:
Frequency and Time Domain Measurements
Pulsed Light Sources (frequency & pulse widths)
Mode-Locked Lasers
ND:YAG (76 MHz) (150 ps)
Pumped Dye Lasers (4 MHz Cavity Dumped, 10-15 ps)
Ti:Sapphire lasers (80 MHz, 150 fs)
Mode-locked Argon Ion lasers
Directly Modulated Light Sources
Diode Lasers (short pulses in ps range, & can be modulated by synthesizer)
LEDs (directly modulated via synthesizer, 1 ns, 20 MHz)
Flash Lamps
Thyratron-gated nanosecond flash lamp (PTI), 25 KHz, 1.6 ns
Coaxial nanosecond flashlamp (IBH), 10Hz-100kHz, 0.6 ns
Modulation of CW Light
Use of a Pockel’s Cell
Pulsed Emission
0
Polished on a side
exit plane
Pockel’s Cell
Mirror
Polarizer
90
Polarizer
Radio Frequency
Input
Double Pass Pockel’s Cell
CW Light Source
The Pockel’s Cell is an electro-optic device
that uses the birefringment properties of calcite
crystals to alter the beam path of polarized light. In
applying power, the index of refraction is changed
and the beam exiting the side emission port (0
polarized) is enhanced or attenuated. In applying RF
the output becomes modulated.
Time Correlated Single Photon Counting
Sample Compartment
Pulsed Light Source
Timing Electronics
or 2nd PMT
Filter or Monochromator
Neutral density (reduce to one photon/pulse)
PMT
Constant Fraction
Discriminator
Photon Counting PMT
TAC
Time-to-Amplitude
Converter (TAC)
Multichannel
Analyzer
Instrument Considerations
Excitation pulse width
Counts
Excitation pulse frequency
Timing accuracy
Time
Detector response time (PMTs 0.20.9 ns; MCP 0.15 to 0.03 ns)
Histograms built one photon count at a time …
1
8
6
4
Fluorescence Decay
Fluorescence
2
0.1
8
6
Instrument Response Function
4
2
0.01
8
6
4
0
50
100
150
200
250
300
Channels (50 ps)
(1) The pulse width and instrument response times determine the time
resolution.
(2) The pulse frequency also influences the time window. An 80 MHz
pulse frequency (Ti:Sapphire laser) would deliver a pulse every 12.5
ns and the pulses would interfere with photons arriving later than the
12.5 ns time.
Polarization Correction
There is still a polarization problem in the geometry of our excitation and
collection (even without a monochromator)!!
Will the corrections never end ???
[1] = I0 + I90
[2] = I0 + I90
[3] = I0 + I90
[4] = I0 + I90
An intuitive argument:
[4]
[6]
0
[1]
Polarized Excitation
[3]
[5] =
[6] =
2 x I90
2 x I90
Total = 4 x I0 + 8 x I90
[2]
0
[5]
The total Intensity is proportional to:
I0 + 2 x I90
90
Setting the excitation angle to 0 and the
emission polarizer to 54.7 the proper weighting
of the vectors is achieved.*
*Spencer & Weber (1970) J. Chem Phys. 52:1654
Frequency Domain Fluorometry
Pockel’s Cell
Sample Compartment
CW Light Source
Filter or Monochromator
RF
PMT
PMT
Turret
Reference
S1
S2
Signal
Locking Signal
Signal
RF
Synthesizers
S1 and S2
Analog PMTs (can also be done with photon counting)
Digital Acquisition
Electronics
S1 = n MHz
S2 = n MHz + 800 Hz
Computer Driven
Controls
Similar instrument
considerations as
With TCSPC
Lifetime Station #3, LDF, Champaign IL, USA
& hiding under the table:
RF Amplifiers
Frequency Synthesizers